U.S. patent number 6,259,524 [Application Number 09/403,635] was granted by the patent office on 2001-07-10 for photobleachable luminescent layers for calibration and standardization in optical microscopy.
This patent grant is currently assigned to The University of Amsterdam. Invention is credited to Godefriedus J. Brakenhoff, Rick I. Ghauharali, Johannes Willem Hofstraat.
United States Patent |
6,259,524 |
Hofstraat , et al. |
July 10, 2001 |
Photobleachable luminescent layers for calibration and
standardization in optical microscopy
Abstract
The invention pertains to a calibration layer comprising an
optically transparent polymer containing an amount of
photobleachable luminscent material present in such a way that the
final polymer film contains less than 10 wt. % of luminophore and
has an optical attenuation of less than 0.3 absorption units in the
wavelength region of interest. The invention further is concerned
with a method of calibration of an optical image device, preferably
an optical or Raman microscope, by using the decrease in
luminescence as the result of photobleaching between two
consecutive images for calibration.
Inventors: |
Hofstraat; Johannes Willem
(Veldhoven, NL), Brakenhoff; Godefriedus J.
(Amsterdam, NL), Ghauharali; Rick I. (Amsterdam,
NL) |
Assignee: |
The University of Amsterdam
(NL)
|
Family
ID: |
8228254 |
Appl.
No.: |
09/403,635 |
Filed: |
January 10, 2000 |
PCT
Filed: |
April 17, 1998 |
PCT No.: |
PCT/EP98/02358 |
371
Date: |
January 10, 2000 |
102(e)
Date: |
January 10, 2000 |
PCT
Pub. No.: |
WO98/49537 |
PCT
Pub. Date: |
November 05, 1998 |
Foreign Application Priority Data
|
|
|
|
|
Apr 25, 1997 [EP] |
|
|
97201235 |
|
Current U.S.
Class: |
356/243.4;
436/8 |
Current CPC
Class: |
G01N
21/278 (20130101); G01N 21/6458 (20130101); G01N
21/65 (20130101); Y10T 436/10 (20150115) |
Current International
Class: |
G01N
21/64 (20060101); G01N 21/65 (20060101); G01N
21/63 (20060101); G01J 001/10 () |
Field of
Search: |
;356/243.1,243.2,243.3,243.4,243.5,243.6 ;436/8,10,19
;250/252.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
LC. Smith et al., "Digital Imaging Fluorescence Microscopy",
IEEE/Seventh Annual Conference of the Engineering in Medicine and
Biology Society, (1985), pp. 967-970..
|
Primary Examiner: Pham; Hoa Q.
Attorney, Agent or Firm: Knoble & Yoshida, LLC
Claims
What is claimed is:
1. A calibration layer comprising an optically transparent polymer
containing a photobleachable luminescent material wherein the
polymer contains less than 10 wt % of luminescent groups
originating from the luminescent material and has an optical
attenuation of less than 0.3 absorption units in the wavelength
region of 250 to 1700 nm.
2. The calibration layer of claim 1 wherein a photobleachable
luminescent group contained in the photobleachable luminescent
material is covalently attached to a sidechain polymer the relative
molar content of which is lower than 10%.
3. A method of calibration of an optical image device by:
a. photobleaching the calibration layer of claim 1 as a series of
images from different parts of the calibration layer;
b. calculating the mean initial luminescence intensity and the mean
bleach rate for each series;
c. calculating a detection efficacy distribution from the data of
b; and
d. using the decrease in luminescence as the result of
photobleaching between two subsequent images as a measure for
calibration.
4. The method according to claim 3 wherein an optical or Raman
microscope is calibrated.
Description
BACKGROUND OF THE INVENTION
The invention pertains to the preparation and use of thin,
photobleachable luminescent layers for calibration and
standardization of optical imaging devices, such as optical or
Raman microscopy. For the quantitative application of optical and
Raman microscopy, it is essential that the intensities in the
images acquired with these microscopic techniques are determined
only by the spatial distribution of the concentration, absorption,
and emission characteristics of the luminophores in the specimen
under investigation. If this is not possible, the image intensities
should at least be proportional to these parameters. Generally,
however, image intensity variations are not only determined by the
specimen, but also by spatial non-uniformities of the optical
system of the microscope across the field of view, so that only
qualitative investigations can be performed. In order to realize
the images required for quantitative microscopy, the microscope
must be calibrated and standardized. The thus obtained images allow
the comparison of different samples obtained on the same
microscope, also at a different point in time, or the comparison of
images obtained on different microscopes, provided that the
different microscopes have been calibrated in the same way.
In earlier work, calibration and standardization of an optical
microscope was attempted by an approach using images of a uniform
luminescent layer (K. R. Castleman, Digital Image Processing.
Prentice-Hall, Englewood Cliffs, N.J., 1979, and Z. Jericevic, B.
Wiese, J. Brayan & L. C. Smith, "Validation of an Image
System," in Luminescence Microscopy of Living Cells in Culture,
Part B, Quantitative Luminescence Microscopy-Imaging and
Spectroscopy, edited by D. Lansing Taylor and Y. Wang, Academic
Press, San Diego, Calif., 1989). Such an approach has three
disadvantages. Firstly, in the case of an image of a luminescent
layer, the product of the illumination and detection efficiency
distributions is measured, and no information on the separate
distributions is available. Secondly, completely uniform
luminescent layers are difficult to obtain. Thirdly, the results of
calibration and standardization based on this approach are affected
by luminescence photobleaching of the layer. For general
calibration and standardization in optical microscopy, it would be
preferable to have an approach which does not suffer from these
disadvantages. Jericevic et al. (Z. Jericevic, B. Wiese, J. Brayan
& L. C. Smith, "Validation of an Image System," in Fluorescence
Microscopy of Living Cells in Culture, Part B, Quantitative
Fluorescence Microscopy-Imaging and Spectroscopy, edited by D.
Lansing Taylor and Y. Wang, Academic Press, San Diego, Calif.,
1989) attempted to do away with the first disadvantage by using
luminescence photobleaching techniques for the determination of
only the illumination distribution. In his method, at least 20
images of a uniform, photobleaching luminescent layer were
required. They showed that by numerically fitting the luminescence
intensity decrease in each pixel of the first image with an
exponential function, it was possible to determine only the
excitation intensity distribution in the field of view of the used
microscope (Z. Jericevic, D. M. Benson, J. Bryan, & L. C.
Smith, "Rigorous Convergence Algorithm for Fitting a
Monoexponential Function with a Background Term Using the
Least-Squares Method," Anal. Chem., 59 (1987), 658-662). There are
several drawbacks to this method and experimental approach.
Firstly, a luminescent layer has to be prepared by spreading an
FITC-IgG mixture on a microscope slide. With such a procedure, it
is very difficult to obtain a uniform luminescent layer. Secondly,
the method provides only the illumination distribution; no
information about the detection distribution is obtained. Thirdly,
the determination of the illumination distribution is based on
numerically fitting routines, which renders the method relatively
slow.
BRIEF SUMMARY OF THE INVENTION
This invention describes the preparation and use of thin,
photobleachable luminescent films for the calibration and
standardization of an optical or Raman microscope in the wavelength
region of 250 nm-1700 nm, preferably 250 nm-1100 nm, and more
preferably 350 nm-900 nm.
DETAILED DESCRIPTION OF THE INVENTION
This is achieved according to the invention by the preparation of a
photobleachable luminescent calibration layer and its subsequent
use for the determination of excitation intensity and detection
efficiency distributions in the field of view of the used
microscope. The term photobleaching comprises all processes which
result in the reduction of the intensity of luminescence light
generated at the wavelength of excitation. Excitation may be done
by laser or by a focused light source in the wavelength ranges
defined above. Examples of such processes are photo-oxidation,
photo-reduction, photo-isomerization or photo-addition reactions,
or light-induced electron transfer processes.
It is sufficient for the effectiveness of the invention that the
prepared calibration layer is approximately uniform, luminescent,
and photobleachable, preferably approximately uniform, luminescent,
and mono-exponentially photobleachable in a certain regime. The
calibration layer should satisfy the following requirements.
(i) The luminescence intensity of luminophore in the calibration
layer should be proportional to the excitation intensity, the
luminophore concentration, and the illumination duration.
(ii) The rate of photobleaching of the luminophore in the
calibration layer should be proportional to the illumination
intensity and independent of the luminophore concentration.
(iii) The luminescence quantum yield, the absorption cross-section,
and the bleach factor--defined as the ratio of the rate of
photobleaching to the excitation intensity--of the luminophore in
the calibration layer should be independent of the
micro-environment within the layer.
The first two requirements already suffice for qualitative
calibration of the measurement. The third requirement in
combination with the first two allows absolute measurement of an
image in optical or Raman microscopy.
The calibration layer is applied on an optically flat and
transparent substrate by spin-coating, dip-coating or rod-coating
(doctor blading) of a, preferably, 1-30 wt % solution of an
optically transparent polymer containing an amount of
photobleachable luminescent material present in such a way that the
final polymer film contains less than 10 wt % of luminophore and
has an optical attenuation of less than 0.3 absorption units in the
wavelength region of interest, or of a solution of a sidechain
polymer with an amount of photobleachable luminescent groups
covalently attached to it, in such a way that the relative molar
content of the sidechains is lower than 10% and the optical
attenuation of the calibration layer is less than 0.3 absorption
units in the wavelength region of interest. The useful
concentration region is determined by the necessity to prevent
intermolecular interactions (energy transfer) and inner filter and
concentration quenching effects, which may lead to deviations tom
simple mono-exponential decay. Optical attenuation more than 0.3
absorption units is possible, but mathematical corrections are
required. Such attenuation is therefore less preferred. Suitable
polymeric materials, which are transparent across the wavelength
region of interest, are polyacrylates, polymethacrylates,
polycarbonates, polyolefins, polyethers, polyurethanes,
polyetherketones, polyesters, polystyrenes, polysiloxanes, and the
like, or copolymers thereof. Suitable polymeric sidechain materials
are based on the same optically transparent building blocks as
applied in the transparent polymer types mentioned above and a
suitable luminescent and photobleachable molecule which is equipped
with a functional group so that it either may be attached to said
polymer or may react with other functional monomers to form a
luminescent sidechain-main chain polymer. Alternatively, thin films
may be prepared by making use of sol-gel glass formation
approaches.
The luminescent material used should be photobleachable, which
means that the intensity of luminescence should be reduced by
illumination in the microscope at the applied excitation
wavelength. A number of light-induced processes may result in such
photobleaching; some examples are photo-oxidation, photo-reduction,
photo-isomerization or photo-addition reactions, or light-induced
electron transfer processes. All luminescent photochromic materials
may be used. The photobleachable luminescent material may undergo
such change either reversibly or irreversibly.
In view of the excellent homogeneity of the luminescent layers
obtained according to the above-mentioned procedure, even the
direct luminescence intensity can be used for calibration.
With the prepared calibration layer, absolute excitation intensity
and detection efficiency distributions in the field of view of the
used microscope can be determined from images, before and after
partial photobleaching, of the calibration layer and the
luminescence quantum yield, the absorption cross-section, and the
bleach factor of the luminophore in the calibration layer, as
follows.
When the luminescence intensity of the calibration layer is
proportional to the excitation intensity, the luminophore
concentration, and the illumination duration, when its
photobleaching is mono-exponential, and when its rate of
photobleaching is proportional to the excitation intensity and
independent of the luminophore concentration, an image of the layer
acquired before any photobleaching has taken place, referred to
below as the "first image," can be written as the product of the
image exposure time, the luminescence quantum yield, the absorption
cross-section, the bleach factor, and the concentration
distribution of the luminophore in the calibration layer, and the
excitation intensity and detection efficiency distributions of the
used microscope. The detection efficiency includes all elements of
the detection pathway important for the conversion of the intensity
to be detected up to the intensity value of a pixel in the final
image, such as the finite collection solid angle of the objective
lens, the reflectivity and transmittance of the optical elements in
the detection pathway, and the quantum efficiency of the detector.
An image, referred to below as the "second image," acquired after
the calibration layer has been bleached during a certain time
interval, can be written as the product of the first image and an
exponential function which is determined by the bleach factor, the
excitation intensity, and the bleach time interval.
Based on these two images the relative excitation intensity
distribution--or a distribution proportional to the excitation
intensity distribution--in the field of view of the used microscope
can be determined by calculating the logarithm of the ratio between
the first and second images of the calibration layer. The absolute
excitation intensity distribution can be determined by calculating
the ratio of the relative excitation intensity distribution and the
bleach factor of the luminophore in the calibration layer to the
bleach time interval. It is important to point out that for the
determination of this--relative or absolute--excitation intensity
distribution, it is not required that the calibration layer is
uniform.
Once the relative excitation intensity distribution has been
determined, the relative detection efficiency distribution--or a
distribution proportional to the detection efficiency
distribution--can be determined as follows. Firstly, a distribution
proportional to the product of the detection efficiency and
luminophore concentration distributions, referred to below as the
"product distribution," is determined by calculating the ratio of
the first image to the relative excitation intensity distribution.
Secondly, a number of product distributions are determined from the
same number of image pairs, first and second images, with each
image pair acquired from a different part of the calibration layer.
By averaging these different product distributions, the
contribution of possible non-uniformities of the luminophore
concentration distribution can be eliminated, and a distribution
proportional to only the detection efficiency distribution, i.e.,
the relative detection efficiency distribution, is obtained. The
number of product distributions required for averaging depends on
the uniformity of the calibration layer: for uniform layers, no
averaging is required, but the less uniform the layer is, the
larger the number of different product distributions should be. For
many applications the determination of the relative distributions
is already sufficient.
When the direct luminescence intensity is used, the excitation
intensity distribution and the detection efficiency distribution
cannot be determined separately. For many applications, e.g.,
shadow correction procedures, it is sufficient to use the product
of the intensity distributions for calibration purposes.
The absolute detection efficiency distribution can be determined by
calculating the product of the relative detection efficiency
distribution, the bleach factor of the luminophore in the
calibration layer, and the bleach time interval, and dividing the
result by the image exposure time and the luminescence quantum
yield, the absorption cross-section, and the mean luminophore
concentration of the luminophore in the calibration layer. The
parameters which have to be known for absolute determination of the
excitation intensity and detection efficiency distributions are the
absorption cross-section, the luminescence quantum yield, and the
bleach factor of the calibration layer. All three parameters can be
determined independent of the microscope used.
The absorption cross-section of the calibration layer at a certain
wavelength can be determined by measuring the optical attenuation
at the same wavelength and combining this information with the
thickness of the layer and its luminophore concentration.
The luminescence quantum yield of the calibration layer can be
determined through comparison of the luminescence of the
calibration layer with the luminescence of a reference layer of
which the luminescence quantum yield is known.
The bleach factor of the calibration layer can be determined by
measuring the relative decrease of the luminescence intensity after
illumination with a known excitation dose, i.e., energy per unit
area.
With the excitation intensity and detection efficiency
distributions known, a number of calibration and standardization
steps in optical microscopy are available.
(i) The method can be employed to compare the excitation intensity
and detection efficiency distributions of different microscopes, or
of the same microscope at different points in time. Differences
between the overall performance of microscopes can be attributed to
the excitation pathway, the detection pathway or both. Such
information can be used to selectively optimize the pathway that
limits the performance. Another possibility is to adjust the
illumination and detection parameters of different microscopes in
such a way that equal--or at least comparable--excitation and
detection conditions result This facilitates the comparison of
measurements in which one (type of specimen is studied either with
different microscopes or with the same microscope at different
times.
(ii) The excitation intensity distribution is important in the
interpretation of the intensity variations in images obtained with
so-called luminescence bleach rate imaging (G. J. Brakenhoff, K.
Visscher & G. Gijsbers, "Luminescence Bleach rate Imaging," J.
Microsc., 175 (1994), 154-161). in that imaging mode, the local
rate of photobleaching rather than the luminescence intensity is
used as a contrast parameter for image formation. Spatial
non-uniformities of the excitation intensity distribution lead
to--apparent--variations of the observed rate of photobleaching.
With an experimentally determined excitation intensity distribution
available, these apparent variations can be corrected.
(iii) The method can be used for the correction of intensity
variations in an image of a specimen under investigation which are
caused by non-uniformities of the optical system of the microscope,
a procedure referred to as "shading correction." In the simple case
of a luminescently labelled specimen of which the detected
luminescence intensity is proportional to both the excitation
intensity and the detection efficiency, shading correction is
accomplished by calculating the ratio of the image of the specimen
under investigation and the product of the--relative or
absolute--excitation intensity and detection efficiency
distributions. The fact that with the method, the excitation
intensity and detection efficiency distributions are available
separately implies that also in more complicated specimens, for
example specimens in which non-linear dependencies occur, shading
correction is possible.
(iv) The method can be employed for the quantitative investigation
of specimens. The intensity variations in a shading corrected image
of a specimen are independent of the microscope used to acquire the
image and are determined only by specimen related factors such as
the concentrations of the luminophores in the specimen and their
absorption and emission characteristics. When shading correction is
based on the absolute excitation intensity and detection efficiency
distributions, the intensities in the shading corrected image of a
specimen can be used to quantitatively determine these specimen
related factors. For example, if a luminophore is available which
can be used to luminescently label a specimen and if the
luminescence quantum yield and the absorption-cross section of this
luminophore are known and independent of the micro-environment
within the specimen, the intensities in the shading corrected image
can be used to quantitatively determine the concentration of this
luminophore in the specimen on a microscopic level.
(v) The excitation intensity and detection efficiency distributions
can be used for active image correction by modulating illumination
and detection parameters during image acquisition in such a way
that a spatially uniform illumination and detection efficiency
results. This possibility is important for bleach rate imaging,
photoactivatable processes, assessment of biological cell damage,
etcetera.
EXAMPLES
To demonstrate the applicability of the invention, a luminescent
and photobleachable calibration layer was prepared and used for
shading correction of images acquired with a confocal luminescence
microscope. The luminescent and photobleachable calibration layer
was based on the luminophore 4-dimethylamino-4'-nitrostilbene
(DANS). Solutions of DANS and polymethylmethacrylate (PMMA) in
chloroform were prepared and used to spin-coat standard glass cover
slips used for optical microscopy. For the investigation of the
influence of the luminophore concentration on the luminescence
intensity and the rate of photobleaching, three calibration layers
were prepared with relative concentrations of 0.2, 0.5 and 1.0.
DANS in PMMA can be excited in the wavelength range <250 nm-550
nm; it fluoresces in the wavelength range 500-850 nm. Upon
irradiation, photobleaching of the fluorophore takes place, mainly
due to photo-oxidation.
The microscope used for this demonstration was an Olympus IMT-2
inverted microscope (Olympus Corporation, Lake Success, N.Y., USA),
equipped with an INSIGHT PLUS bilateral confocal line scanning unit
(Meridian Instruments Inc., Okemos, Mich., USA) and a 100.times.,
NA=1.32, oil immersion objective lens. Luminescence was excited at
488 nm, using an air cooled Argon ion laser (model 532, Omnichrome,
Chino, Calif., USA). The excitation intensity could be varied by
insertion of neutral density filters (NDFs) in the laser delivery
path of the microscope. A total of four NDFs were available, with a
transmittance ranging from 1% to 50%. The generated luminescence
was detected with a cooled CCD camera (model DDE/3200, Astromed,
Cambridge, UK) through a long-pass filter with a cut-off wavelength
at 520 nm. A Hewlett-Packard model 725/50 workstation
(Hewlett-Packard, Palo Alto, Calif., USA) was used for image
collection and exposure control via a mechanical laser shutter.
Image analysis was carried out on the same workstation, using the
image processing package Scillmage (T. K. Ten Kate, R. van Balen,
A. W. M. Smeulders, F. C. A. Groen & G. A. de Boer, "SCILIAM, a
Multi-level Interactive Image Processing Environment," Pattern
Recognition Letters, 11 (1990), 429-441).
For the investigation of the photobleaching characteristics of the
calibration layer, so-called "bleach curves" were determined by
acquiring a series of images--a time series--from a certain part of
the calibration layer. The image exposure time was the same for all
images in the time series, and no additional exposure occurred
between successive images. From each time series, two quantities
were determined: the mean initial luminescence intensity and the
mean bleach rate. The mean initial luminescence intensity was
calculated by averaging the intensities in the first image of the
time series. The mean bleach rate should ideally be determined by
averaging the bleach rates calculated for each pixel in the first
image of the time series for a number of regions of interest
(ROIs), which were chosen arbitrarily in the image. The data for
each individual ROI were fitted with a (mono-) exponential
function. The mean bleach rate was obtained as the mean of the
individual ROI bleach rates.
For the luminescent layer it has been verified that its
luminescence and photobleaching characteristics conform to the
requirements of the method, i.e., the luminescence intensity in the
first image of the calibration layer--or the initial luminescence
intensity--should be proportional to the excitation intensity, the
luminophore concentration, and the image exposure time, its
photobleaching should be mono-exponential, and its rate of
photobleaching should be proportional to the excitation intensity
and independent of the luminophore concentration.
It was found that the conditions for the luminescence
characteristics are satisfied by the proposed calibration layer.
The photobleaching of the calibration layer initially was not
strictly mono-exponential; however, close to mono-exponential
photobleaching of the calibration layer could be realised by
"pre-bleaching" the layer. The data obtained after 180 sec of
pre-bleaching were used to calculate "the rate of photobleaching,"
which appeared to be proportional to the excitation intensity and
independent of the luminophore concentration in the layer.
Therefore, after suitable pre-bleaching, the requirements for the
photobleaching characteristics are fulfilled by the prepared
calibration layer.
The excitation intensity distribution can be determined from two
images of the calibration layer, which are separated by a time
interval in which the calibration layer is partially bleached. To
calculate the relative excitation intensity distribution, the first
and second images were acquired with a pre-bleaching time of 180
sec and a bleach time interval of 150 sec, since in this
time-interval the decrease of the luminescence intensity is
described well with a mono-exponential function. In these images, a
stripe- and spot-like pattern can be seen, which is independent of
the part of the calibration layer from which the images were
acquired. This indicates that the stripe- and spot-like pattern is
caused by non-uniformities of the optical system of the microscope.
Inspection shows that the excitation intensity is not distributed
uniformly over the image region, but that a stripe-like pattern
occurs. It was found that this pattern is caused by the dichroic
mirror in the microscope. The relative magnitude of the variations
in excitation intensity over the image region can be expressed as
the coefficient of variation (CV)--the ratio of the standard
deviation to the mean. The measurement shows that in the
relative--and absolute--excitation intensity distribution,
variations of approximately 10% occur over the image region. The CV
is a measure of the variation across the entire image. It should be
pointed out that locally much larger variations can occur.
The detection efficiency distribution can also be determined from
two images--the first and second images--of the calibration layer.
The relative detection efficiency distribution is determined from
the product distribution, which is proportional to the product of
the detection efficiency and luminophore concentration
distributions. The product distribution is determined from the
first image of the calibration layer and the already determined
relative excitation intensity distribution. By calculating the
product distribution from different, randomly chosen, parts of the
calibration layer and averaging the results, the relative detection
efficiency distribution is obtained. By averaging a number of
product distributions, measured at different parts of the
calibration layer, the relative detection efficiency distribution
is obtained. As already noted, this stripe-like pattern is caused
by the dichroic mirror in the microscope, and since this mirror is
part of both the excitation and detection pathway, the same pattern
is visible in the relative excitation intensity and detection
efficiency distributions. Also visible are dark spots which cannot
be seen in the relative excitation intensity distributions. These
are probably caused by small dust particles or other irregularities
in the detection pathway of the microscope. The relative magnitude
of the variations in detection efficiency can be estimated by again
taking the CV as a measure of the variations. Variations of
approximately 25% occurred in the relative--and absolute--detection
efficiency distributions. Again, locally the variations can be much
larger.
With the known relative excitation intensity and detection
efficiency distributions shading correction of an image of a
specimen can be carried out by calculating the ratio of the image
of the specimen to the product of the relative excitation intensity
and detection efficiency distributions.
Comparison before and after correction indicates a clear reduction
of the intensity variations. Also visible is that a calibration
layer feature--the deliberately photobleached line-shaped
region--is well preserved after the correction procedure, whereas
the intensity variations caused by the non-uniformities of the
optical system have disappeared. A different way is to visualize
the effect of the shading correction, in which case the intensities
are plotted before and after correction. It is clear from this that
the intensity variations after correction are significantly smaller
than before correction. The effect of the shading correction was
quantified by calculating the CV of the intensities in the images.
The results show CVs of approximately 22% and 4% before and after
correction, respectively. This means that the shading correction
procedure achieved a more than five-fold decrease of the intensity
variations. Locally the decrease of the image intensity variations
obviously will be much larger.
* * * * *